Fig. 8. Hybridization pattern of P. purureum DNA digested
with isoschizomers to detect methylation of eKn I
families. Total DNA from P purpureum (PI300086) was
digested with either Eco RII or Bst NI,
electrophoresed in 2% agarose, Southern blotted,
probed at 650 C with nick translated P. qlaucum IB23
140 bp Kpn I monomers, and washed at 650 C in 0.07X
SSPE and 0.1% SDS. All lanes contained 2 ug of DNA.
Bands of hybridization which migrate at 560 bp and
980 bp are labeled. Lane 1 is Eco RII digested DNA.
Lane 2 is DNA digested along with the DNA in lane 1,
extracted, precipitated, and redigested with Eco
RII. Lane 3 is Bst NI digested DNA. Lane 4 is DNA
digested along with the DNA in lane 3, extracted,
precipitated, and redigested with Bst NI.

1 2

S8980 bp

560 bp

34

53

I repeat families to the extent that Bst NI did. The

restriction endonuclease isoschizomers used here test for

methylation at the trinucleotide CNG, N being any nucleotide.

This experiment indicates that the KDn I families of tandemly

arrayed repetitive sequences within the P. purpureum nuclear

genomes are heavily methylated.

The percentage of the purpureum genotype PI300086

(Fig. 9, panel A) and P. glaucum Tift 23 (Fig. 9, panel B)

genomes comprised of the Kpn I repeat families was determined

along with copy numbers per cell. Various amounts of either P.

purpureum 140 bp and 160 bp Kpn I monomers or P. glaucum 140

bp KEn I monomers were used to create a standard curve. The

nuclear DNA of either species was then compared to the

standard curve created for each of the species. A dot blot

apparatus was used to facilitate the binding of samples to the

membrane for probing. The dot blot for the P. purpureum

samples was probed with 140 bp and 160 bp Kpn I monomers from

P. purpureum. The dot blot for the P. glaucum samples was

probed with 140 bp Kpn I monomers from pearl millet. For

napiergrass and pearl millet, 100 nanograms of nuclear DNA

(lane 1) was compared to various amounts of monomers; the

various amounts were 5, 10, 15, 20, 25, and 30 nanograms in

lanes 2-7 respectively. For P. purpureum genotype PI300086,

16.4 +/- 1.5 percent of the nuclear genomes are composed of

Kpn I repetitive element hybridizing sequences. The P. glaucum

genotype Tift 23 has 19.9 +/- 0.8 percent of the nuclear

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genome composed of KDn I repetitive element hybridizing

sequences. Reassociation kinetics have indicated that about

20% of the pearl millet genome is comprised of the highly

repetitive class of sequences (Patankar et al. 1985); the 140

bp &pn I tandemly arrayed repetitive sequences are thus the

major highly repetitive DNA sequences in P. glaucum. The KDn

I families of P. purpureum are likely the major repetitive

sequence families within the nuclear genomes as well. Based

upon a mole of nucleotide pairs averaging 618 grams, one

picomole of DNA equals 9.74 X 108 bp. P. purpureum nuclear DNA

is approximately 5.78 picograms (Taylor and Vasil 1987). Thus

there are 6.2 X 106 copies of &Kn I repeats per cell. P.

glaucum, which contains approximately 4.9 picograms per cell

(Patankar et al. 1985), has 6.8 X 106 copies of Kpn I repeats

per cell.

To compare the average sequences (Grellet et al. 1986) of

the 140 bp and 160 bp Kpn I families of P. purpureum

(PI300086), P. glaucum Tift 23, P. squamulatum (PS26), and P.

hohenackeri (PS156), either four or five individual KDn I

monomers isolated from individual bands from a 3%

polyacrylamide gel were sequenced. The 140 bp Kpn I average

sequences from P. squamulatum and P. glaucum are compared to

the P. purpureum average sequence in Fig. 10. This is sequence

data from one individual plant of each species. Where there is

an n in the P. purpureum or P. squamulatum average sequences

the bases of two of the four sequences at that position were

Fig. 10.

Comparison of the average sequence (Grellet et al.
1986) of P. urureum (PI300086) to the average
sequences of P. suamulatum (PS26) and P. glaucum
Tift 23. Average sequences of P. purpureum and P.
squamulatum are composed of sequences of four
individual Kpn I family monomers. The P. alaucum
average sequence is composed of five individual
sequences. The three sequences are aligned for
comparison purposes. The P. squamulatum and P.
glaucum sequences are compared to the P. urpureum
average sequence. Differences between the sequences
are designated by an asterisk above the position of
the base difference in the sequence being compared
to the P. purpureum average sequence. In the P.
purpureum and P. suamulatum average sequences the
appearance of an n is due to their being two bases
equally represented at that position; if one of
these two bases is the same as the base at the
identical position in a sequence being compared then
it is not considered a difference between the two
sequences.

Fig. 11. Comparison of the average sequence (Grellet et
al. 1986) of P. ourureum (PI300086) to the
average sequences of E. scuamulatum (PS26) and E.
hohenackeri (PS156). All average sequences are
composed of four individual KEn I family monomers.
Also shown in this figure are sequences from two
individual Epn I family monomers from P. setaceua
(PS247) and EP. villosum (PS249); differences
between these two sequences are represented as an
n with the two bases below the n being from the
two sequences. The sequences are aligned so that
the insertion and the deletion present in the P.
hohenackeri average sequence can be compared to
the other average sequences. Differences between
the average sequences are designated by an
asterisk above the position of the base difference
in the average sequence being compared to the P.
purpureum average sequence. In the average
sequences the appearance of an n is due to their
being two bases equally represented at that
position; if one of these two bases is the same as
the base at the identical position in a sequence
being compared then it is not considered a
difference between the two sequences. All four
individual sequences were different at position 48
in the e purpureum average sequence. Bases from
position 130 to 148 are underlined in all six
sequences. This 18 bp underlined region represents
the major difference between the 140 bp and 160 bp
En I family sequences.

Subrepeats within the P. purpureum average 160
bp Kpn I sequence were aligned according to
conserved regions present in these subrepeats. These
conserved regions present in the subrepeats are
underlined below each subrepeat. Below the EP
purpureum subrepeats is a maize satellite DNA
monomer segment (Peacock et al. 1981). The regions
of similarity between the P., urpureun
subrepeats and the maize satellite DNA monomer
segment are underlined below the maize sequence. The
maize sequence is aligned such that the positioning
of the conserved regions between the species can be
compared to the conserved regions from the
subrepeats.

Aligned Subrepeats Within the Pennisetum purpureum Average
160 bp Kon I Sequence; Similarity to a Maize Repeat is
Also Shown